siRNA / miRNA gene silencing Rat Brain endothelial cells HIF-1α

Get tips on using TRIzol™ LS Reagent to perform RNA isolation / purification Tissue - human brain tissue

Get tips on using Isolate II RNA Mini Kit to perform RNA isolation / purification Tissue - Mouse Brain

Get tips on using DC™ Protein Assay Kit I to perform Protein quantification Tissue - mouse brain

Get tips on using pSilencer™ 4.1-CMV neo to perform shRNA gene silencing Human - SiHa AEG-1

Get tips on using AM5770: pSilencer™ 3.1-H1 neo to perform shRNA gene silencing Mouse - CT26 OPN

Get tips on using GeneChip® HT 3' IVT PLUS Reagent Kit to perform Microarray RNA amplification & Labeling - Mouse brain tissue Biotin

Get tips on using LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells to perform Live / Dead assay mammalian cells - rat nucleus pulposus

Get tips on using LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells to perform Live / Dead assay mammalian cells - rat primary hepatocytes

Get tips on using GATA-1 shRNA Plasmids (h) to perform shRNA gene silencing Human - TF‐1 GATA‐1

Reporter gene assays are designed to test the regulation of the expression of a gene of interest. This is usually done by linking the promoter of the gene of interest with a gene such as a firefly luciferase, which can be easily detected by addition of luciferin that leads to an enzymatic reaction to produce luminescence. The enzymatic reaction can be correlated to the expression of the gene of interest. Another luciferase gene that can be used is Renilla luciferase. For an appropriate luciferase assay: 1. the reporter should express uniformly in all cells, 2. specifically respond to effectors that the assay intends to monitor, 3. have low intrinsic stability to quickly reflect transcriptional dynamics. It is important to have an equal number of cells plated in each testing condition to avoid any incorrect readouts. Reporter assays could be single or dual reporter assays. The reporter could be both luciferases. Most dual-luciferase assays involve adding two reagents to each sample and measuring luminescence following each addition. Adding the first reagent activates the first luciferase reporter reaction; adding the second reagent extinguishes first luciferase reporter activity and initiates the second luciferase reaction. Dual-luciferase assays have some advantages, including 1. reduces variability, 2. reduces background, 3. normalizes differences in transfection efficiencies between samples.